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J Biol Chem. 2018 Mar 16;293(11):4026-4036. doi: 10.1074/jbc.RA117.001642. Epub 2018 Jan 30.

Structure-function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism.

Author information

1
From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
2
the Université Grenoble Alpes and CNRS, CERMAV UPR 5301 601, rue de la chimie, 38000 Grenoble (France) and Institut de Chimie Moléculaire de Grenoble, ICMG, FR-CNRS 2607, Grenoble, France.
3
the Institute for Nanotechnology and Advanced Materials, and Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel, and.
4
From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada, miroslaw.cygler@usask.ca.
5
the Department of Biochemistry, McGill University, Montreal, Quebec H3G 0B1, Canada.

Abstract

Ulvan is a major cell wall component of green algae of the genus Ulva, and some marine bacteria encode enzymes that can degrade this polysaccharide. The first ulvan-degrading lyases have been recently characterized, and several putative ulvan lyases have been recombinantly expressed, confirmed as ulvan lyases, and partially characterized. Two families of ulvan-degrading lyases, PL24 and PL25, have recently been established. The PL24 lyase LOR_107 from the bacterial Alteromonadales sp. strain LOR degrades ulvan endolytically, cleaving the bond at the C4 of a glucuronic acid. However, the mechanism and LOR_107 structural features involved are unknown. We present here the crystal structure of LOR_107, representing the first PL24 family structure. We found that LOR_107 adopts a seven-bladed β-propeller fold with a deep canyon on one side of the protein. Comparative sequence analysis revealed a cluster of conserved residues within this canyon, and site-directed mutagenesis disclosed several residues essential for catalysis. We also found that LOR_107 uses the His/Tyr catalytic mechanism, common to several PL families. We captured a tetrasaccharide substrate in the structures of two inactive mutants, which indicated a two-step binding event, with the first substrate interaction near the top of the canyon coordinated by Arg320, followed by sliding of the substrate into the canyon toward the active-site residues. Surprisingly, the LOR_107 structure was very similar to that of the PL25 family PLSV_3936, despite only ∼14% sequence identity between the two enzymes. On the basis of our structural and mutational analyses, we propose a catalytic mechanism for LOR_107 that differs from the typical His/Tyr mechanism.

KEYWORDS:

His/Tyr mechansim; Polysaccharide lyase; Ulvan lyase; carbohydrate processing; catalysis; crystallography; mutagenesis; polysaccharide; structural biology; substrate specificity

PMID:
29382716
PMCID:
PMC5857984
DOI:
10.1074/jbc.RA117.001642
[Indexed for MEDLINE]

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